Background: Flexion-rotation test predominantly measures rotation in C1-2 segment. Restriction in flexion-rotation may be due to direct limitation in C1-2, but also to a premature tightening of the alar ligament as a result of lack of movement in C0-1 or C2-3. The aim of this study was to compare the effect of a 20-min single cervical exercise session, with or without manual therapy of C0-1 and C2-3 segment in flexion-rotation test, in patients with chronic neck pain and positive flexion-rotation test. Methods: Randomized controlled clinical trial in 48 subjects . Range of motion and pain during flexion-rotation test, neck pain intensity and active cervical range of motion were measured before and after the intervention. Results: Significant differences were found in favour of the manual therapy group in the flexion-rotation test: right (p < 0.001) and left rotation (p < 0.001); pain during the flexion-rotation test: right (p < 0.001) and left rotation (p < 0.001); neck pain intensity: (p < 0.001); cervical flexion (p < 0.038), extension (p < 0.010), right side-bending (p < 0.035), left side-bending (p < 0.002), right rotation (p < 0.001), and left rotation (p < 0.006). Conclusions: Addition of one C0-C1 and C2-C3 manual therapy session to cervical exercise can immediately improve flexion-rotation test and cervical range of motion and reduce pain intensity. Mobility restrictions of the upper cervical spine have been associated with neck pain and headaches ,2. Most Although it is considered a test to assess the rotation of the C1-2 segment, the presence of painful joints in the lower cervical spine reduced the available range in the flexion-rotation test to 37.5° . This inTakasaki et al.  recruiteSome studies have investigated the effectiveness of treating upper cervical spine restriction in the flexion-rotation test with joint mobilization and manipulation. These studies used joint manipulation and mobilization that was applied directly to the C1-2 segments at the end of the cervical rotation ROM ,18. ThisTwo previous studies have shown that the mobilization of the C0-1 segment produced an improvement in flexion-rotation test . HidalgoHowever, there are no studies that have analysed the immediate effect of the manual therapy treatment of the C0-1 and C2-3 segment to improve the mobility in a restricted flexion-rotation test, without treating the C1-2 segment.Cleland et al. suggest in different articles ,24,25 thFinally, cervical exercise is effective in reducing pain  and incrOur hypothesis was that a one-session treatment of segments C0-1 and C2-3 can immediately improve the flexion-rotation test. The aim was to compare the effect, in the flexion-rotation test, of a 20-min session of manual therapy in C0-1 and C2-3 segment accompanied by cervical exercise, with the effect of a 20-min session of cervical exercise solely, in patients with chronic neck pain and positive flexion-rotation test.www.random.org). The randomization results were placed in a concealed opaque envelope, and participants were assigned to intervention groups.A prospective, longitudinal, randomized (simple 1:1), and controlled assessor-blind clinical trial was conducted. A researcher not involved in the study randomized the intervention for each patient using computer software . This study complied with the ethical principles for research on human beings as per the Declaration of Helsinki . It was approved by the local ethics committee (Comité Ético de Investigación Clínica en Aragón “CEICA”) in its Records n°13/2018 on 4 July 2018. All participants were required to provide written and informed consent to participate. This study followed all CONSORT criteria.The study was carried out in the facilities of the University of Zaragoza, Spain  were included in the study. The inclusion criteria comprised: medical diagnosis of chronic neck pain with more than three months of evolution , a positThe primary outcome measure in this study was the ROM in the flexion-rotation test. Secondary outcomes measures were neck pain intensity, pain intensity during the flexion-rotation test, and cervical ROM. The methodology proposed by Hall et al.  was follFor current neck pain intensity at the time of evaluation, the NPRS was used  . NPRS haActive cervical ROM was measured in all cardinal planes for the assessment of cervical mobility. For active mobility testing, patients were asked to sit straight ahead, and to move their head as far as they could without pain in the different cardinal planes of movement . A CROM A researcher specialized in manual therapy, with more than 5 years’ experience in physical therapy, performed the measures before and after the intervention. This researcher was blinded to the allocation group of each patient throughout the process. Intervention was carried out by a different researcher with more than 5 years’ experience in physical therapy. The intervention was provided individually in the facilities at the University of Zaragoza. Participants in both groups received a single 20-min session.After baseline assessments, patients began the cervical exercise. They were taught to perform the contraction of deep neck flexor muscles (longus capitis and longus colli) using the Stabilizer Pressure Biofeedback Unit  in supine ,46 Figu. The exeIt is assumed that this altered recruitment pattern is a contributing factor to the chronification of neck pain. Training deep neck flexor muscles achieves the improvement of neuromuscular coordination and not of the strength and endurance, so a single session can obtain changes in the motor pattern .The Exercise group carried out one 20-min session, composed of 2 blocks of 10 repetitions. They held each repetition for 10 s , and hadThe Manual Therapy+Exercise group (MT + E group) carried out a 20-min session of treatment. The manual therapy techniques were applied depending on the clinical findings in each patient, and the objective was to restore the mobility of C0-1 and C2-3 segments before applying cervical exercises. Manipulation (high-velocity low amplitude) and/or mobilization (low-velocity high amplitude) techniques of C0-1 and C2-3 segments were applied ,49 Figu. ManipulAll the manipulation and mobilization techniques used in this trial follow IFOMPT recommendations to reduce the risk of adverse events .p > 0.05). Outliers were examined. No value was excluded because extreme values did not cause any significant bias. Within- and between-group differences were analyzed using repeated measures of ANOVA and one-way ANOVA. If the assumption of sphericity was violated, the Greenhouse-Geisser correction was utilized for interpretation [p < 0.05.Statistical analysis was conducted with the SPSS 23.0 package . There was no loss of follow-up in the study. The mean, confidence interval and standard deviation were calculated for each variable. The Kolmogorov–Smirnov test was used to determine a normal distribution of quantitative data  with a mean age of 51.9 (14.5) years old met all eligibility criteria and agreed to participate. Then, 24 participants were randomly assigned to each group, received their assigned treatment, and were analyzed for intention-to-treat . Enrollmp < 0.001; d = 1.14) and left rotation  ROM. A statistically significant decrease in NPRS was also observed during both rotations ; left NPRS rotation ).No statistically significant changes were found in the within-group analysis  for the p < 0.001; d = 1.72) and left rotation  ROM, and in right NPRS rotation  and in left NPRS rotation .In the between-group analysis , statistp < 0.001; d = 1.41).In the within-group analysis  for the p < 0.001; d = 1.30).In the between-group analysis , statistp < 0.008; d = 0.37), extension , and left side-bending . In the MT + E group, a significant increase was found only in left side-bending .In the within-group analysis  for exerp < 0.038; d = 0.62), extension , right side-bending , left side-bending , right rotation , and left rotation .In the between-group analysis , statistThis study found an immediate increase in ROM on the flexion-rotation test in the MT + E group. The improvement in the flexion-rotation test observed in the MT + E group was superior to the minimal detectable change (4.7° for right rotation and 7.0° for left rotation) described by Hall et al. . This grExcept for the C1-2 segment, there is little information regarding the effects of the treatment of other cervical segments on upper cervical rotation ROM examined with the flexion-rotation test .There is evidence that neck pain, ROM, and disability may be improved by treatment of segments and spinal regions remote from the symptomatic segment . HidalgoA possible explanation for these results in the MT + E group in our study may be due to the relationship between the articular, muscular, and ligamentous tissue between C0-1, C1-2 and C2-3 segments  dysfunctIn our study, we found a significant reduction in NPRS for both rotations during the flexion-rotation test in the MT + E group. There is only one previous study that records NPRS during the flexion-rotation test , but theA significant decrease in NPRS was found in MT + E group. In contrast, the exercise group did not experience any statistically significant change in neck pain intensity. Several studies found immediate improvements with manual therapy and exercise ,55, withBialosky JE et al.  exposes A significant increase in the left side-bending was found in the MT + E group. In contrast, we found a significant decrease in the cervical ROM in the exercise group in flexion, extension, and left side-bending movements.The measuring inaccuracy in the side-bending of the CROM range is between 5° and 10° . In thisOne study explains that a full ROM in the upper cervical spine is needed to be able to properly contract the deep flexor muscles . It is pFinally, the results of this study support the hypothesis that mobilization and manipulation of C0-1 and C2-3 is an effective treatment for restoring upper cervical spine rotation measured by the flexion-rotation test. These results are consistent with other studies examining the effect of mobilization and manipulation treatments targeting the C1-2 segment . HoweverThe main limitation of this study was that only the immediate effects of the techniques were measured. Future studies with short-, mid-, and long-term follow-ups in this group of patients with a positive flexion-rotation test using these treatment techniques within a multimodal approach  would be necessary.An important limitation was the possible representativeness of this subgroup of chronic neck pain patients with positive flexion-rotation test, and the external validity among all the possible subgroups of patients suffering from this syndrome.Another significant limitation was that we do not know what specific intervention has had the most significant impact on the patients. Different studies have shown good effects treating the C0-1 segment , but we Finally, the measuring inaccuracy of the CROM range is between 5° and 10° . TherefoIt would be necessary to undertake more studies with this subgroup of patients with chronic neck pain and positive flexion-rotation test to understand their behavior.This study showed that one 20-min manual therapy session of C0-1 and C2-3 segments with exercise can immediately improve flexion-rotation test and pain intensity in patients with chronic neck pain and positive flexion-rotation test.


Adipose-derived mesenchymal stem cells (AD-MSCs) are a type of stem cell that is abundant and widely used. The molecular characteristics of AD-MSCs from different passages from donors of different ages have not been well elucidated.Six kinds of AD-MSCs  with E denoting cells derived from an elderly patient, Y denoting cells derived from a young patient, and 1, 2, and 3 representing passages 3, 6, and 10) were obtained from human abdominal adipose tissue. We obtained the protein expression profile, the mRNA expression profile, the lncRNA expression profile, and the methylation profile of each kind of AD-MSC by sequencing. After calculating the stemness indices, genes related to stemness were extracted. The multiomics correlation analysis was performed in the stemness-related genes. In addition, short time-series expression miner (STEM) analysis was performed for all cell passages and donor ages. To further explore the biological functions of the stemness-related genes, we performed Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. Finally, the lncRNA-KEGG network and transcription factor (TF)-KEGG network were constructed based on the RNAInter database and TRRUST v2 database.The stemness of the Y1, E1, and Y2 cells was higher than that of the E2, Y3, and E3 cells. The stemness was the highest for Y1 cells and the lowest for E3 cells. STEM analysis showed that five stemness-related gene clusters were associated with the cell passages, and only one gene cluster was associated with age. The enrichment analysis results showed that the biological processes (BPs) and KEGG pathways were mainly involved in the proliferation, differentiation, and migration of cells. The global regulatory landscape of AD-MSCs was constructed: 25 TFs and 16 lncRNAs regulated 21 KEGG pathways through 27 mRNAs. Furthermore, we obtained a core stemness-related gene set consisting of ITGAV, MAD2L1, and PCNA. These genes were expressed at higher levels in Y1 cells than in E3 cells.The multiomics global landscape of stemness-related gene clusters was determined for AD-MSCs, which may be helpful for selecting AD-MSCs with increased stemness. When used for different therapeutic procedures aimed at treating various forms of tissue damage, the autologous fat graft is indeed a solution. Recent studies have shown that adipose-derived stromal vascular fractions (AD-SVFs) , 2 and aSome studies have shown that platelet-rich plasma (PRF) and hyaluronic acid (HA) dressings alone or in combination are also helpful to promote tissue repair –12. PRF The stemness of AD-MSCs is mainly reflected in their ability to maintain self-renewal, cell differentiation, and proliferation . A previThis study attempts to elucidate the multiomics and molecular characteristics of AD-MSCs derived from donors of different ages and from different cell passages. We found that the stemness of AD-MSCs from younger donors was increased. In addition, the stemness of early-passage (passage 3) AD-MSCs was the highest compared with that of intermediate-passage (passage 6) and late-passage (passage 10) AD-MSCs. These findings may provide guidance on how to select AD-MSCs with increased stemness, as well as whether AD-MSCs should be injected directly or post-expanded.g. Then, we added collagenase solution  to the AT, which was shaken gently in a 37 °C water bath to digest the adipose tissue. The digested AT was centrifuged again for 10 min and then filtered to remove the debris at 180g. Next, erythrocyte lysis buffer was added to the cell pellet , which was resuspended and centrifuged for 10 min at 180g. The stromal vascular fraction was plated and cultured in tissue culture-treated flasks in adipose-derived stem cell plating medium  and 1% penicillin/streptomycin. After overnight incubation, the nonadherent cells were removed, and the medium was changed to fresh medium. After 20 to 48 h, the cells were expanded until they were nearly confluent to obtain the passage 0 AD-MSCs. The expansion rate and morphology of the AD-MSCs were observed under a microscope. When the AD-MSCs were at a confluence of 80% to 90%, they were passaged at a ratio of 1:3. The cell number of passage 0 AD-MSCs was 1 × 106 and 2 × 106 in young patient and the elderly patient, respectively. When amplifying passages 3, 6, and 10, the cell number was 1 × 108, 1 × 1010, and 2 × 1012 in the young patient. While in the elderly patient, the cell number was 1 × 108, 5 × 109, and 2 × 1011 when amplifying passage 3, 6, and 10. Subsequently, 1 × 108 cells from each kind AD-MSCs were collected for sequencing. AD-MSCs from the two patients were harvested at three time points during amplification for multiomics analysis to obtain the early-passage (passage 3), intermediate-passage (passage 6), and late-passage (passage 10) cells. A total of 6 types of AD-MSCs  were obtained; E represents the elderly patient, Y represents the young patient, and 1, 2, and 3 represent passages 3, 6, and 10. The workflow of this study is shown in Fig. The study was performed with the permission of the ethics committee of the First People’s Hospital of Nanning, Guangxi Zhuang Autonomous Region. We obtained written informed consent from donors. Human lipoaspirate from the abdominal subcutaneous tissue of healthy females (one young patient who was 27 years old and an elderly patient who was 63 years old) was stored for less than 48 h at 4 °C before processing. The adipose tissue (AT) was gently aspirated using 10-ml Luer-Lok syringes and then placed in a tube, which was centrifuged for 3 min at 1200All RNA was isolated from AD-MSCs by TRIzol purification (Invitrogen), and genomic DNA was removed with the help of gDNA eliminator columns from the RNeasy Mini Kit (Qiagen). In addition, spectrophotometry  was used to assess the RNA quality along with a Bioanalyzer (Agilent). The RNA was enriched for mRNA  by poly(A) isolation, and the mRNA was fragmented and subjected to first-strand cDNA synthesis. After second-strand cDNA synthesis, the double-stranded cDNA was quantified and purified. The library was constructed by using a protocol from Illumina with the NEBNext DNA Sample Prep Reagent kits (NEB). The double-stranded cDNA was end-repaired, and the adapters were ligated to the DNA fragments; poly(A) tracts RNA sequences were then added. After size selection (200~600 bp) and UDGase treatment to ensure strand specificity, the adapter-modified DNA fragments were amplified by PCR. Next, 76-base paired-end sequencing was performed on an Illumina instrument.The strand-specific paired-end reads were screened for ribosomal RNA by alignment against known rRNA sequences (RefSeq) using Bowtie software . The rRN2(HpaII/MspI) value represented methylation and was analyzed as a continuous variable. For most loci, each fragment was categorized as either hypomethylated if the log ratio was greater than zero or methylated if the centered log HpaII/MspI ratio was less than zero. The average methylation level for all CpG islands in a gene was considered the methylation level of the corresponding gene in the present study.LCM-DNAs from AD-MSCs were fragmented into 100–500 bp fragments. Then, the genomic DNA was extracted from the AD-MSCs using a high-salt procedure, and the HELP assay was performed as previously described. The assay involved interrogating the cytosine methylation status and performing comparative isoschizomer profiling on a genomic scale. The genomic DNA from AD-MSCs was digested by HpaII (a methylcytosine-sensitive enzyme) and MspI, and then the MspI- and HpaII-produced fragments were amplified through PCR mediated by ligation. Both of the amplified fractions were submitted to Roche-NimbleGen, Inc. . Then, these fractions were labeled and hybridized with a human hg17 custom-designed oligonucleotide array (50-mers) covering 25,626 HpaII-amplifiable fragments (HAFs), which were located at gene promoters. HpaII-amplifiable fragments are defined as genomic sequences. Each fragment on the array was represented by 15 individual probes distributed randomly throughout the microarray slide. The microarray covered 50,000 CpGs corresponding to 14,000 gene promoters. The signal intensities were calculated as the robust mean of their component probe-level signal intensities after intensive quality control. The logg for 20 min. The concentration of the protein was measured by a quantitative protein kit .Each sample was homogenized with 5 volumes of glass sand. Then, 200 μl radioimmunoprecipitation assay (RIPA) buffer containing phenylmethylsulfonyl fluoride (PMSF) was added to lyse the cells. Then, the samples were sonicated. All soluble proteins were collected after centrifugation at 13,400g. The ultrafiltration cartridge was centrifuged in 0.25 M TEAB (triethylammonium bicarbonate). The protein pellets were reconstituted in 0.25 M urea/50 mM TEAB and digested with 2% trypsin overnight (Promega).One hundred micrograms of protein was reduced, alkylated, and precipitated by the chloroform precipitation method to digest the protein. First, 55 mM Reducing Reagent  was added to the proteinase and incubated at 60 °C for 60 min. Then, the protein was mixed with Cysteine Blocking Reagent . After deionized water and 70% ethanol were added to the 10 KD ultrafiltration cartridge, the protein solution was poured into the ultrafiltration cartridge. The solution was centrifuged for 20 min at 13,4004, pH 10) and mobile phase A . The flow for peptide elution was set to a rate of 20 μl/min. The fractions were collected and acidified with trifluoroacetic acid (50%). The fractions were vacuum-dried prior to LC-MS/MS. The fractions were dissolved in buffer and pelleted. The supernatant was loaded onto analytical columns and identified with a Q Exactive system . The components of the mobile phase used for LC-MS were formic acid (0.1%) and 80% CAN. The flow rate of the analytical columns was set at 350 nl/min. The peptides were analyzed by using a 3-step gradient for 65 min. The parameters of the first-grade MS were a maximum injection time of 40 ms and a resolution of 70,000. The scan range was from 350 to 1800 m/z. The second-grade MS spectra were acquired at a resolution of 17,500. The top 20 precursors were selected for each MS cycle.A high-performance liquid chromatography (HPLC) system  was used to perform strong cation exchange (SCX) fractionation chromatography. The isobaric tags for relative and absolute quantification (iTRAQ)-labeled peptides were separated with a linear gradient formed by mobile phase B . Then, to obtain the mRNA/lncRNA expression profiles, the data were normalized using the “voom” function of the limma package [The raw counts of the mapped reads were aggregated using featureCounts . The gen package  in R. ThStemness is considered to represent the potential for differentiation and self-renewal of the cell of origin, which possesses the ability to produce all cell types in the adult organism . The TCGr) was calculated using the Hmisc package [P < 0.01 and r > 0.9 were considered to indicate a strong correlation. The correlation among coding gene (mRNA) DNA methylation, protein expression, and corresponding mRNA expression and the long noncoding gene (lncRNA) DNA methylation and corresponding lncRNA expression were also explored.The correlation between stemness and mRNA/long noncoding RNA (lncRNA) expression/protein expression was used to identify mRNAs/lncRNAs/proteins that are highly related to stemness. The correlation between stemness and mRNA/lncRNA/DNA methylation was also explored. The Pearson correlation coefficient  biological processes (BPs) and KEGG pathways were visualized with a bubble diagram. GSVA [ClueGO plug-in [Cytoscape software [P value adjusted by the false discovery rate < 0.05 was considered significant.To explore the biological functions of the stemness-related gene profiles, Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analyses were performed using the  package  in R. Tham. GSVA  was used plug-in  in Cytossoftware . The GO Interactions between stemness-related lncRNAs and their target genes were downloaded from the RNAInter database . The intA total of 19,189 mRNAs, 21,965 lncRNAs, and 3205 proteins were included in these analyses. The DNA methylation levels of 8070 mRNAs and 4308 lncRNAs were measured. Stemness was closely associated with the cell passage of the AD-MSCs in the following order of decreasing association: early passage > intermediate passage > late passage  that comprise a core stemness-related gene set. As shown in previous studies, MAD2L1, PCNA, and ITGAV are related to the proliferation and differentiation of cells. ITGAV is a kind of mRNA, and its product belongs to the integrin α chain family, which serves as a major receptor for differentiation and cell proliferation . MAD2L1 Our study presents several limitations. First, the study is based on bioinformatics prediction; therefore, further experimental verification is needed. In addition, the sample size involved in this study was small, and we need to verify the results in a larger data cohort later.The multiomics global landscape of stemness-related gene clusters was constructed for AD-MSCs. Moreover, we identified a core stemness-related gene set comprised of ITGAV, PCNA, and MAD2L, which may be helpful for selecting AD-MSCs with increased stemness.Additional file 1 : Table S1. The 555 genes belonging to the 5 stemness-related gene clusters associated with cell passages.Additional file 2 : Table S2. The thirty-five genes belonging to the single stemness-related gene cluster associated with age.Additional file 3 : Table S3. GO_BPs enriched in adipose-derived mesenchymal cells. GO: gene ontology, BPs: biological processes.Additional file 4 : Table S4. KEGG pathways enriched in AD-MSCs. AD-MSCs: adipose-derived mesenchymal cells.


The hazard of implant loss for implants of non-smokers was 2.92 times higher in the mandible compared to the maxilla (p = 0.01). The overall mean bone loss was 0.97 mm  at the implant level and 0.90 mm  at the patient level. Smokers lost significantly more bone compared to non-smokers in the maxilla (p = 0.024) but not in the mandible. Only the maxilla showed a significant difference in the probability of implant success between smokers and non-smokers . Taking jaw into account, implants in smokers experienced a 2.6 higher risk of developing peri-implantitis compared to non-smokers (p = 0.053). Conclusion: Dental implants with a fluoride-modified surface provided a high 10 years’ survival with limited bone loss. Smokers were, however, more prone to peri-implant bone loss and experienced a higher rate of implant failure, especially in the upper jaw. The overall bone loss over time was significantly higher in smoking patients, which might be suggestive for a higher peri-implantitis risk. Hence, smoking cessation should be advised and maintained after implant placement from the perspective of peri-implant disease prevention.Background: The purpose of this study was to compare the survival and peri-implant bone loss of implants with a fluoride-modified surface in smokers and non-smokers. Material and Methods: All patients referred for implant treatment between November 2004 and 2007 were scrutinized. All implants were placed by the same surgeon (B.C.). The single inclusion criterion was a follow-up time of at least 10 years. Implant survival, health, and bone loss were evaluated by an external calibrated examiner (S.W.) during recall visits. Radiographs taken at recall visits were compared with the post-surgical ones. Implant success was based on two arbitrarily chosen success criteria for bone loss (≤1 mm and ≤2 mm bone loss after 10 years). Implant survival in smokers and non-smokers was compared using the log-rank test. Both non-parametric tests and fixed model analysis were used to assess bone loss in both groups. Results: A total of 453 implants in 121 patients were included for survival analysis, and 397 implants in 121 patients were included for peri-implant bone-loss analysis. After a mean follow-up time of 11.38 years , 33 implants out of 453 initially placed had failed in 21 patients, giving an overall survival rate of 92.7% and 82.6% on the implant and patient level, respectively. Cumulative 10 years’ survival rate was 81% on the patient level and 91% on the implant level. The hazard of implant loss in the maxilla was 5.64 times higher in smokers compared to non-smokers ( One in five adults in the world smokes tobacco, despite the fact that negative effects on oral and general health are well known. Every seventh death in the world 13%) was the result of direct smoking in 2017; a further 2% was the result of secondhand smoke. This means 15%—close to 1-in-6 deaths—was the result of tobacco. However, since 1990, there is a declining global trend in smoking reflected almost everywhere across the world 3% was th. SmokingOver the last decade, implants surfaces have been modified from smooth/rough to moderately rough surface texture, expressed by an average Sa value of 1–2 µm ,34,35. TThe purpose of the present study was to evaluate the 10 years’ survival and success of implants with a fluoride-modified surface in smokers and non-smokers treated under daily clinical and non-specifically selected conditions.TM, Dentsply, Astra Tech, Mölndal, Sweden), following the manufacturer’s guidelines and suturing. Implant installation was immediately followed by radiographs (baseline) with commercially available film-holders  using the parallel long-cone technique to visualize marginal bone-to-implant contact points and implant threads. Care was taken to shoot perpendicular on the implant axis. The individualization of standard film holders was not manageable in private practice. If implants threads were not clear, the radiograph was discarded, and a new radiograph was taken. To determine marginal bone levels correctly, the digital images were magnified by the software. Possible distortions were solved by calibration, based on the known abutment height and implant diameter. There might be a slight difference in the horizontal plane, but the evaluated implants were round, so this difference should be rather limited.All patients in need of implant placement between November 2004 and 2007 were evaluated. During intake and at the 10-year follow-up session, a medical history was taken, including self-reporting of smoking habits. The initial 2-years report was presented previously . The samHence, bone loss beyond the reference point was reported from the time of surgery, and initial bone remodeling was included in the total bone level changes over time. After the final restorations were made by the referring dentist, a professional maintenance schedule (including radiographic follow-up) was proposed to each patient, whereby the frequency was based on the clinical situation and individual needs. Given the fact that the patients were referred by and, therefore, returned to their original dentist, only patients that maintained their visits at the specialist clinic were included in the current study. These patients were prospectively followed up for at least 10 years. Briefly, this consisted of a recall interval of 6 or 12 months during the first 2 years and 12 or 24 months during the following years. All implants with at least 10 years of follow-up and part of the professional maintenance recall system of the specialist center were included to evaluate implant survival and peri-implant bone loss. An independent external examiner (SW) from the University of Ghent performed the recall consultation at the 10 years’ follow-up and had access to the patient files. All patients were thoroughly informed and signed a written consent form. The study protocol was approved by the ethical committee of the Ghent University Hospital  under number B670201524796.Smoking was defined as the smoking of at least 1 cigarette a day and was based on self-reporting. Ex-smokers and non-smokers were combined into the group of non-smokers. A history of periodontitis was based on the following criteria: (a) radiographic proof of bone loss extending 33% of the root length of residual teeth at the time of referral; (b) patients who were treated with (non)surgical periodontal treatment before implant therapy; (c) when before implant treatment, hopeless teeth were extracted due to periodontitis; (d) edentulous patients with evidence of periodontitis at the time of referral based on radiographs obtained in retrospect from the referring dentist. Peri-apical radiographs were analyzed with the use of digital Software  with an accuracy of 0.1 mm. These were taken from the day of surgery up to at least 10 years in function. The crestal bone level was calculated at both mesial and distal sites of each implant by measuring the distance between the reference point (lower border of the smooth implant collar) to the first marginal bone-to-implant contact .Bone loss beyond the reference point was calculated by comparing peri-apical radiographs taken during recall visits after 3 months, 1, 2, and 10 years with baseline . The mean of both bone level readings  was calculated to obtain a single value per implant. Plaque- and bleeding assessment was performed at six sites . Pocket n = 121). For survival analysis at the implant level, we only included implants from patients who had at least one implant with ≥ 10 year-follow-up and with known observation time (n = 453). Kaplan–Meier estimates of implant survival at the patient level were compared between smokers and non-smokers with the log-rank test. The estimated survival rates at 1, 2, 5, and 10 years were reported together with the 95% confidence intervals, which were calculated using the “log-log” approach. Hazard estimates of implant loss at the implant level were compared between smokers and non-smokers, overall and per jaw using the Robust Score test for a simple Cox proportional hazards model. Robust standard errors were estimated to take into account the clustering of implants within patients. The estimated survival rates at 1, 2, 5, and 10 years were reported together with the 95% confidence intervals—calculated using the “log-log” approach. These confidence intervals didn’t take into account the clustered design. A multiple Cox proportional hazards model—for smoking status, jaw, and their two-way interaction—was fitted with robust estimation of the standard error to take into account the clustering of implants within patients. Robust Wald 95% confidence intervals and corresponding p-values were reported. Life tables  based on a two-way random model with absolute agreement. The observed mean, standard deviation, minimum, and maximum were used to describe bone loss beyond the reference point in several subgroups at different time points. Cumulative frequencies of bone loss in mm were plotted for different time intervals. Lower curves would have a smaller proportion of bone loss <2 mm than higher curves and, hence, more bone loss. Cumulative frequencies of bone loss in mm were plotted for smokers and non-smokers. For the analyses at the implant level, a linear mixed model for bone loss in mm was fitted with a random intercept for the patient to account for multiple implants within a patient and with smoking status, jaw, and their two-way interaction as fixed effects. Estimated marginal means at the original target scale for smoking status and for smoking status * jaw were requested together with the pairwise comparisons. No test was performed to compare mean bone loss at the patient level between smokers and non-smokers because the residuals were not normally distributed, and, unlike with the analysis at the implant level, one could not solely rely on the central limit theorem due to the smaller sample size. A non-parametric test would compare the mean rank between smokers and non-smokers, instead of comparing the actual mean.Implant success was defined in two ways: Firstly, as ≤1 mm bone loss after 10 years, and, secondly, as ≤2 mm bone loss after 10 years. For analysis at the implant level, a generalized linear mixed model with a binomial distribution and logit link for implant success was fitted with a random intercept for the patient and with smoking status, jaw, and their two-way interaction as fixed effects. Estimated marginal means at the original target scale for smoking status and for smoking status * jaw were requested together with the pairwise comparisons. For the analyses at the patient level, Fisher’s exact test was used to test for a difference in the proportion of implant success between smokers and non-smokers.Mean bleeding on probing  and mean probing pocket depth were calculated. The possibility of a statistically significant difference was examined by non-parametric testing (Mann–Whitney U test). Peri-implant mucositis was identified as the presence of bleeding and/or suppuration on gentle probing with or without increased probing depth compared to previous examinations and absence of bone loss beyond crestal bone level changes resulting from initial bone remodeling. Peri-implantitis was defined as loss of crestal bone with time ≥3 mm, suppuration, and/or bleeding on probing, with or without increasing probing pocket depth ≥6 mm . A gener®, Armonk, NY, USA).Statistical descriptive analysis and non-parametric testing for patient compliance were performed using SPSS v23 .Of the original 300 patients included in the previous report , 81 patients had never been maintained in the specialist clinic and had returned to their own dentist for regular maintenance, 6 maintained patients had passed away, and 72 had ignored maintenance over time at the specialty clinic and returned to their referring dentist or indicated not to participate in the proposed recall program. In total, 141 patients were cooperative and compliant with the maintenance program and were invited for the research assessment, and 121 responded positively (drop-out 14.2%). Forty-eight were male, and 73 were female, with a mean age of 65.2 years . An overview of the distribution of implant length and diameter, with notification of implant loss, is shown in p = 0.001).Of the total of 121 patients, 43 had single crowns, 51 had fixed partial dentures, 24 had fixed cross-arch bridges, 2 had overdenture on locators, and 1 patient had an overdenture on a bar-structure. On the implant level, 67 implants supported single crowns, 180 supported fixed partial dentures, 200 supported fixed cross-arch bridges, 4 supported overdentures on locators, and 2 implants supported an overdenture on a bar-structure. Only one patient, a non-smoker, had diabetes (regulated with medication), and one patient started oral bisphosphonates during follow-up, several years after implant treatment. Smokers showed significantly higher compliance compared to non-smokers , 33 implants out of 453 initially placed had failed in 21 patients. An absolute survival rate of 92.7% and 82.6% on the implant and patient levels was seen, respectively. The cumulative 10 years’ survival rate (CSR) was 81% on the patient level and 91% on the implant level .Eleven out of 76 implants failed in smokers, and 22/377 in non-smokers, resulting in absolute survival rates of 85.5% and 94.2%, respectively. CSR’s were 82% vs. 75% on the patient level and 93% vs. 81% on the implant level for non-smokers and smokers, respectively. Eight implants failed before prosthetic loading, all in non-smokers.p = 0.047). However, the hazard of implant loss for implants of the maxilla was 5.64 times higher in smokers compared to non-smokers (95% CI for the HR went from 1.82 to 17.5) (p = 0.003). The hazard of implant loss for implants of non-smokers was 2.92 times higher in the mandible compared to the maxilla (95% CI for the HR went from 1.29–6.62) (p = 0.01).Regarding the jaw of treatment, 17/272 (6.25%) implants in the upper jaw and 16/181 (8.84%) implants in the lower jaw failed. For smokers, 3/35 (8.57%) implants failed in the mandible and 8/41 (19.51%) in the maxilla. For the non-smoking group, implant failure for the mandible was 13/146 (8.90%) and for the maxilla 9/231(3.9%). CSR’s in respect of smoking status and jaw are mentioned in p = 0.0031) with smokers having a higher mean bone loss   found no statistical difference between the three treatment protocols . From th1.12 mm) .p = 0.006). The difference in bone loss between maxilla and mandible was not significant within both groups (smoking p = 0.47 and non-smoking p = 1) ) versus 0.80 mm ) was found for the maxilla. For the mandible, this was 1.28 mm ) versus 0.78 mm ). Only for the maxilla, the difference of mean bone loss was significant between smokers and non-smokers (g p = 1) .Implant success was calculated using a threshold for individual total bone loss arbitrary set at ≤1 mm and ≤2 mm changes. This was based on additional bone loss measured between 24 and 120 months. p = 0.049, adjusted for jaw). There was a significant difference in the probability of implant success between smokers and non-smokers in the upper jaw . In our sample, a significant difference in the probability of implant success between smokers and non-smokers in the lower jaw was absent . When the criterion was defined as “bone loss ≤2 mm after 10 years of follow-up”, non-smokers showed an overall success rate of 88.7% versus 69.4% for the smoking group, not statistically significant . A significant difference in the probability of implant success was seen in the upper jaw . In our sample, one could not find a significant difference in the probability of implant success in the lower jaw . Only the smoking group showed a significant difference in implant success between maxilla and mandible, with higher implant success in the mandible . There was an indication of effect modification of smoking by the jaw, although not statistically significant . We found no difference in the proportion of implant success at the patient level between smokers and non-smokers .With the success criterion of “bone loss ≤1 mm after 10 years of follow-up”, non-smokers showed 81.2% implant success versus 59.7% for smokers. This difference was significant (p = 0.332). Overall mean pocket probing depth was 4.25 mm  being 4.69 mm  for smokers versus 4.19 mm  in non-smokers (p = 0.086). The overall mean bleeding on probing was 0.30 , with 30% of the implants showing bleeding on probing, 0.29  in non-smokers versus 0.35  in smokers (p = 0.053). When comparing jaws, 22.2% and 11.6% of the implants placed in the mandible experienced peri-implantitis in smokers and non-smokers, respectively. Regarding the upper jaw, this was 34.4% versus 9.8%, respectively. These differences were found to be statistically non-significant (p = 0.228 and p = 0.127). Similarly, no statistically significant difference was present between maxilla and mandible in each group (p = 0.481 for smokers and p = 0.757 for non-smokers).When taken jaw into account, implants placed in patients with smoking habits experienced a 2.6 higher risk in developing peri-implantitis compared to the implants placed in non-smokers. This difference was borderline non-significant  and the meta-analysis by Strietzel and coworkers (2007) ,71. SmokBone loss beyond the reference point was calculated on the patient level because of the systemic character of smoking and on the implant level to avoid masking clinical complications in the case of multiple implants within the same individual. An overall mean bone loss was found of 0.97 mm (SD 1.79) on the implant level and 0.90 mm (SD 1.39) on the patient level after an average of 11.38 years of function. Bone level changes of moderately rough implants are reported to be in the order of 0.18–1.8 mm in the long-term ,63,64,76Smoking has been identified as a predictor of (late) peri-implant bone loss ,55. The Whereas smoking is strongly identified as a risk factor in the literature for periodontal pathology, inconclusive evidence exists regarding smoking as a risk factor or risk indicator for peri-implantitis ,80. SomeImplants adapted with a fluoride-modified surface provided a high 10 years’ survival with limited bone loss. Smokers were, however, more susceptible to peri-implant bone loss and experienced a higher rate of implant failure, especially in the upper jaw. The overall bone loss over time was significantly higher in smoking patients, which might be suggestive for a higher peri-implantitis risk. Hence, smoking cessation should be advised and maintained after implant placement from the perspective of peri-implant disease prevention.


Cryptomeria japonica, Cupressaceae) to apply them in the improvement of protocols for efficient induction of embryogenic cell lines (ECLs). The results of several years of experiments including studies on the influence of initial explant, seed collection time, and explant genotype as the main factors affecting SE initiation from male-fertile, male-sterile, and polycross-pollinated-derived seeds are described. Initiation frequencies depending on the plant genotype varied from 1.35 to 57.06%. The best induction efficiency was achieved when seeds were collected on mid-July using the entire megagametophyte as initial explants. The extrusion of ECs started approximately after 2 weeks of culture, and the establishment of ECLs was observed mostly 4 weeks after extrusion on media with or without plant growth regulators (PGRs). Subsequently, induced ECLs were maintained and proliferated on media with PGRs by 2–3-week-interval subculture routines. Although, the initial explant, collection time, and culture condition played important roles in ECL induction, the genotype of the plant material of sugi was the most influential factor in SE initiation.This study aimed to obtain information from several embryogenic cell (EC) genotypes analyzing the factors that affect somatic embryogenesis (SE) initiation in sugi ( Somatic embryogenesis (SE) is currently the most efficient technique for large-scale propagation in clonal forestry and a powerful tool for plant regeneration in basic biological studies, genetic engineering, and implementation of multi-varietal forestry ,2,3. Howms1/ms1) and an elite tree  heterozygous for MS1 (Ms1/ms1) [MS1 is the most representative male sterility gene found so far in pollen-free individuals of sugi [Sugi is the most important forestry species in Japan, representing 44% of the total reforested area. However, more than 30 million people  suffer from sugi pollinosis, a serious social and public health problem. As a countermeasure against sugi pollinosis, the use of male-sterile plants  is a recommended alternative. Currently, MSPs of sugi are produced mostly by artificial crossing between a male-sterile tree (Ms1/ms1) . The MS1 of sugi . The sel of sugi ,17. Usin of sugi . In this of sugi . On the  of sugi , there iHere, we report the results of several years of experiments on SE initiation in sugi, including studies on the influence of initial explant, seed collection time, and explant genotype, as the main factors affecting SE initiation from male-fertile, male-sterile, and polycross-pollinated-derived seeds. This study aimed to obtain information from several seed families or crosses and to analyze the factors that affect the initial stage of SE process in sugi.2 = 12.829, df = 3, p < 0.05 for mid-June, and p < 0.1 for late-July), but no significant differences were observed between the late-June and early-July collection times. Although the best response for both culture media was recorded in mid-July , for further SE initiation experiments, media supplemented with 10 μM 2,4-dichlorophenoxyacetic acid  and 5 μM 6-benzylaminopurine (BA) were used.The first experimental approach was to determine the effect of plant growth regulators (PGRs) and seed collection times on SE initiation. The entire megagametophyte containing the zygotic embryo at different developmental stages was used as an initial explant. The extrusion of ECs A startedmid-July , germinamid-July C. This rEven though different initial culture media and seed sources were used, the highest SE initiation frequencies in sugi (20–35%) with seeds collected in mid-July was also reported by Ogita et al. . Similar2 = 141.92, df = 2, p < 0. 001) and seed families  except for the “Taga 4” and “Tsukuba 2” families. As widely known, the choice of appropriate explant is a determining factor for successful SE initiation. In this way, the entire megagametophyte preferably from immature seeds was the most common explants reported in conifers [As shown in conifers ,32,33,34conifers . AlthougMS1 at different seed collection times are shown in 2 = 477.1, df = 4, p < 0.001) and seed collection times  with the exception of collection in late-July  was achieved with megagametophytes, whereas the lowest rate (0.46%) was recorded when whole seeds were used as initial explants. The results of the statistical analysis indicated that the proportion of explants with SE initiation response significantly differed among initial explant types  and seed families . The induction of ECs from seeds stored at 5 °C for 1 and 4 weeks was achieved but with no improvement in SE initiation frequencies was observed compared to non-stored megagametophyte explants  comparable with whole seed explants (0.46–4.17%). This result can be attributed to the improper cutting technique applied, which possibly did not allow the extrusion of ECs. More effort is needed to improve this technique to save energy and time in preparing the initial material.2 = 28.997, df = 1, p < 0.001) and among seed collection times . Similar to the results obtained in the experiments with male-fertile and male-sterile-derived seed explants, the best responses from polycross families were recorded with plant materials collected in mid-July  were used as the initial explant for SE initiation in sugi. As shown in mid-July . These r2 = 618.55, df = 2, p < 0.001), it is important to note that the best average rate obtained with polycross-pollinated-derived seeds were the results of only two years of collection. In addition, SE initiation frequencies from male-fertile, male-sterile, and polycross-pollinated-derived seeds showed great variation ranging from 1.35–67.46%  and Ibaraki Prefectural Government Forestry Technology Center  from 1997 to 2011 . To deteMS1 or MS2 [MS1 were tested  and nine or ten parents of mixed pollen (9 mix or 10 mix) in a seed orchard at the Niigata Prefectural Forest Research Institute  from 2019 to 2020 . The effw/v available chlorine) sodium hypochlorite solution for 15 min and then rinsed three times with sterile distilled water for 5 min each time.After isolation from collected cones, the seeds were surface sterilized with 1% . The pH was adjusted to 5.8 prior to autoclaving the medium for 15 min at 121 °C. Media without PGRs but containing 2 g L−1 activated charcoal  were also tested to compare SE initiation frequencies with those media containing PGRs  and cultured in darkness at 25 °C. The initiation medium containing basal salts reduced to half the concentration from the standard EM medium  was supping PGRs . For all−1 sucrose, 1.5 g L−1 glutamine, and 3 g L−1 gellan gum. Clumps of embryogenic cells (12 per plate) were cultured in darkness at 25 °C.Induced ECs were subcultured every 2–3 weeks on maintenance/proliferation medium containing basal salts reduced to half the concentration from the standard EM medium  supplemep-values were adjusted with a Bonferroni correction [The proportion differentiation of the explants with SE initiation response among seed families, seed collection times, and initial explant types were examined using Pearson’s Chi-squared test. To further elucidate which part of the data was causing the significant differentiation, the residuals of the Chi-squared test were used to conduct the post hoc analysis and the rrection . Pearsonrrection , and therrection .Our research in sugi proved that, although SE initiation was possible from mid-June to late-July, the best induction efficiency was achieved when seeds were collected in mid-July. The best collection time for SE initiation was confirmed in experiments with male-fertile, male-sterile, and polycross-pollinated-derived seed explants. Notwithstanding differences regarding SE initiation frequencies among families observed throughout our experiments, the optimal collection time for almost all seed families was determined around mid-July. Similarly, as reported for other conifers, the megagametophyte explant was also the best plant material for SE initiation in sugi. However, even though the initial explant, collection time, and culture condition played important role in ECL induction, the genotype of the plant material of sugi was the most influential factor in SE initiation. More effort is necessary to obtain experimental information about the SE initiation performance of sugi genotypes using control-pollinated families to select the most appropriate female and male parents. Emphasizing this point, we believe that the polycross pollination technique can be a practical tool for this purpose.


N-acetylcysteine (NAC) and disodium 2,4-disulfophenyl-N-tert-butylnitrone (HPN-07) as a therapeutic for addressing blast-induced neurodegeneration, we measured a significant treatment effect on preservation or restoration of IHC ribbon synapses, normalization of ABR wave V/I amplitude ratios, and reduced behavioral evidence of tinnitus in blast-exposed rats, all of which accorded with mitigated histopathological evidence of tinnitus-related neuropathy and maladaptive neuroplasticity.Tinnitus, the phantom perception of sound, often occurs as a clinical sequela of auditory traumas. In an effort to develop an objective test and therapeutic approach for tinnitus, the present study was performed in blast-exposed rats and focused on measurements of auditory brainstem responses (ABRs), prepulse inhibition of the acoustic startle response, and presynaptic ribbon densities on cochlear inner hair cells (IHCs). Although the exact mechanism is unknown, the “central gain theory” posits that tinnitus is a perceptual indicator of abnormal increases in the gain  of the central auditory system to compensate for peripheral loss of sensory input from the cochlea. Our data from vehicle-treated rats supports this rationale; namely, blast-induced cochlear synaptopathy correlated with imbalanced elevations in the ratio of centrally-derived ABR wave V amplitudes to peripherally-derived wave I amplitudes, resulting in behavioral evidence of tinnitus. Logistic regression modeling demonstrated that the ABR wave V/I amplitude ratio served as a reliable metric for objectively identifying tinnitus. Furthermore, histopathological examinations in blast-exposed rats revealed tinnitus-related changes in the expression patterns of key plasticity factors in the central auditory pathway, including chronic loss of Arc/Arg3.1 mobilization. Using a formulation of  Tinnitus has been linked to common sequelae of acoustic overexposures –3. TinniThe study of the underlying mechanisms that drive tinnitus has increased exponentially over the past several decades, and the prevailing view has shifted to a belief that tinnitus, even when triggered by peripheral cochlear damage, originates within the central auditory system –12. ThisAs such, peripheral auditory damage represents a primary risk factor for the generation of tinnitus, where inner hair cell (IHC) deafferentation, rather than outer hair cell (OHC) loss, is posited to play a key role in driving the disorder –19. AnimHowever, the identification of objective electrophysiological measurements that effectively define and capture evidence of this neurological phenomenon have proven difficult to develop. Auditory brainstem responses (ABRs) in tinnitus subjects have been extensively investigated with the hopes of finding possible abnormalities related to the putative neuropathology, however a consistent objective signature has proven challenging to resolve –28. PrevN-acetylcysteine (NAC) combined with 2,4-disulfophenyl-N-tert-butylnitrone (HPN-07) have demonstrated that the two drugs act synergistically to decrease oxidative stress, neuroinflammation, apoptosis, ischemia-reperfusion injury, and neurodegeneration throughout the auditory pathway [As prior work has revealed that IHC deafferentation represents a logical catalyst for tinnitus, potential therapeutic approaches for their treatment would seemingly be best addressed by preserving or regenerating these peripheral synaptic connections. Oxidative stress is considered to be one of the main causes of neural damage after acoustic injury. Previous studies have shown that peripheral nerve injury induces the production of reactive oxygen species and nitric oxide in axotomized neurons . In addi pathway –43. BaseThe goals of the present study were five-fold: (1) to survey central auditory structures of mild blast-exposed rats for histological evidence of chronic tinnitus-related neuropathy/neuroplasticity; (2) to validate the blast model for the generation of a chronic tinnitus percept; (3) to reveal the peripheral factors that represent likely triggers for tinnitus; (4) to establish a regression equation for predicting tinnitus; and (5) to test therapeutic effects of NAC combined with HPN-07 on addressing behavioral, physiological, and histopathological evidence of tinnitus.Here, we demonstrate that, in animals with behavioral evidence of tinnitus, deafferentation of cochlear nerve fibers triggers compensatory neural plasticity in the central auditory pathway, resulting in the development of tinnitus. Our results revealed that NAC/HPN-07 therapy reduced the risk of tinnitus by approximately 50%, wherein cochlear ribbon synapse preservation or repair and normalization of the expression patterns of homeostatic gating factors in the central auditory pathway were apparently involved. Our regression analyses suggest that ABR wave-V/I amplitude ratios can serve as an objective and reliable tinnitus metric and can provide evidence of treatment-mediated normalization of aberrant central plasticity changes.All procedures regarding the use and handling of animals were approved by the Oklahoma Medical Research Foundation Institutional Animal Care and Use Committee. Eighty Long–Evans male rats, aged 1–3 months, were used in histologic, diagnostic, and therapeutic studies on blast-induced pathology and tinnitus. Rats weighing 250–300 g were obtained from Harlan Laboratories and were housed in the Oklahoma Medical Research Foundation (OMRF) animal care facility. All animals were evaluated by the attending veterinarian upon arrival and were monitored daily by a trained staff of laboratory animal technicians. Animals were maintained on a normal day/night cycle at 21°C with free access to food and water, where following the usual routine, they were provided a 1-week or more acclimation period prior to experimentation. For the electrophysiological, behavioral, and ribbon synapse studies, rats were divided into three groups of twenty: (1) unexposed control group; (2) nontreatment group, in which animals underwent blast exposure and received vehicle treatment with saline; and (3) treatment group, in which animals were subjected to the same blast exposure and received therapeutic treatment with NAC/HPN-07 dissolved in saline solution. For the spiral ganglion neuron and brain biomarkers studies, an independent group of twenty rats were divided into the same experimental cohorts and were subjected to blast and subsequent vehicle or NAC/HPN-07 treatment as described above. All procedures regarding the use and handling of animals were approved by the OMRF Institutional Animal Care and Use Committee.et. al. (2011), whereby the cross-section of the interior of the upper end of the tube increased proportionately to the square of the axial distance [Various blast simulators used in laboratory settings have been developed to generate blast overpressure waves that closely resemble a classic Friedlander wave, with a positive phase and negative phase above and below atmospheric pressure, respectively . Here, odistance . The shoFollowing anesthesia with 90 mg/kg ketamine and 9 mg/kg xylazine, animals were placed inside the shock tube in a rostro-cephalic orientation towards the blast source and were exposed to a single blast wave of 10 psi peak overpressure. Only the head of each rat was subjected to blast exposure, whereas its body was protected from blast injury via a small circular polyvinylchloride holding tube (6″ long with inner diameter of 2″), which was mounted on rails on a metal frame that allowed for precise positioning of a rat along the X-, Y- and Z-axis to ensure that each animal was positioned at a point where the time-pressure profile of the blast wave formed a 10 psi shock front that corresponded to a Friedlander wave. Each 360-400g rat fit snuggly within the holding tube, with only its head exposed to the base of the pinna. Blast wave profiles were verified to maintain consistent exposure pressures between subjects. The overpressures were measured inside of the shock tube by means of a high-frequency piezo-electronic pressure transducer , while the data acquisition and analyses were performed with a PicoScope 3000 oscilloscope .NAC was purchased as a 20% solution from Hospira, Inc. HPN-07 was synthesized and provided at greater than 98.5% purity by APAC Pharmaceuticals, LLC. A mixture of both drugs was prepared in saline to reach a final concentration of 60 mg/mL for each drug. Drug therapy was administered intraperitoneally (i.p.) at 5 mL/kg, starting at 1h after blast exposure and then continuing twice daily for the following two days. Animals in the vehicle control group (which we refer to as the vehicle group in the present study) were injected i.p. with the same volumetric ratio of saline (5 mL/kg) as the active drug formulation, according to the same schedule as the treatment group.AR-α1 , or mouse anti-GluR2  overnight at room temperature. After washing with PBS/T, either biotinylated goat anti-rabbit IgG or horse anti-mouse IgG  was applied to the slides for one hour at room temperature, and Vectastain ABC and DAB kits  were used for the immunolabeling visualization. Immuno-positive cells exhibited a brown reaction product at the sites of the target epitopes. Methyl green was used for nuclear counter-staining. Negative controls were prepared by omitting the primary antibodies. For dual immunofluorescence evaluations of GABAAR-α1 and GAD67, sections were blocked as described above and then incubated with mouse anti-GAD67 antibody  and rabbit anti-GABAAR-α1 (1:500) overnight at room temperature. After washing with PBS, the sections were incubated with appropriate Alexa Fluor® 488 and 568 secondary antibodies  for two hours at room temperature followed by DAPI nuclear labeling and mounting in anti-fade medium. Images were collected with a Zeiss LSM-710 confocal microscope. Antigen retrieval  was employed for GluR2 immunostaining, as well as in GAD67 and GABAAR-α1 dual immunolabeling.Animals used for brain and cochlear sectioning and subsequent immunohistological evaluations in each experimental group (6–8 rats/group) were euthanized and intracardially-perfused with saline followed by 4% paraformaldehyde in 0.1 M phosphate-buffered saline  at nine weeks post-blast. Brains, brainstems and cochleae were removed and post-fixed in the same fixative (one week for brain tissues and overnight for cochleae), washed with PBS, and then stored in PBS at 4°C. The brain and brainstem from each animal were cryoprotected in 30% sucrose in PBS at 4°C until the tissue settled to the bottom of the container, at which time they were embedded in Tissue-Tek  and serially sectioned in a coronal plane with a Thermo Cryotome  at 20 μm. One section out of every ten from each brain and brainstem was mounted onto a gelatin pre-coated slide ; the distance between two adjacent sections on each slide was about 200 μm. Sections were blocked in 1% bovine serum albumin  and either 1% normal horse serum or 1% normal goat serum in PBS, and permeabilized in 0.2% Triton X-100 in PBS (PBS/T). Blocked and permeabilized sections were then incubated with either rabbit anti-Arc , rabbit anti-GABAmedial), the middle third (middle) and the lateral third  sections. In the inferior colliculus (IC), images were collected from the central nucleus of the IC. In the auditory cortex (AC), images were collected from all layers . A modified two-dimensional quantification method was employed to count immuno-positive cells in these nuclei or regions [2) was calculated and statistically analyzed.For quantitative evaluations, images were collected with a BX51 Olympus microscope. In the brain, dorsal cochlear nucleus (DCN) images were collected from the medial third , fixed cochleae were washed with PBS and then decalcified for two weeks in 10% EDTA with solution changes two times each week. Cochleae were then dehydrated, embedded in paraffin, and sectioned in a paramodiolar plane at a thickness of 6 μm. Every 10th section was mounted on a slide , and the mounted sections were processed for immunohistochemical analyses (as detailed below). Cochlear sections were de-paraffinized in xylene and re-hydrated in serial concentrations of ethanol and distilled water. The sections were blocked with 1% BSA and 1% normal goat serum in PBS for 1 hour and then incubated with rabbit anti-VR1 antibody  overnight at room temperature followed by visualization processing as detailed above. Images were collected from the spiral ganglion in the basal and middle turns of all sections on each slide. The number of VR1-positive neurons was quantified using ImageJ software. The percentage of VR1-positive neurons in the SG  was calculated and statistically analyzed.2 rise-fall envelope at frequencies of 4, 8, 16, and 24 kHz. The stimuli were delivered to the ear in free-field mode by a TDT MF1 speaker placed 10 cm lateral to the animal’s pinna. The MF1 was driven by a TDT stereo power amplifier. Electrical brainstem responses to tone bursts were recorded using sub-dermal needle electrodes located at the vertex (active), the ipsilateral mastoid (reference) and the contralateral mastoid (ground), where the responses were amplified 5,000 × before digitization, band-pass filtered from 0.3 to 3 kHz, and averaged across 512 repetitions at a rate of 21/s. Sound intensities were varied in 5-dB steps up and down to identify ABR threshold. The threshold was defined as the lowest dB stimulus level that yielded a repeatable, clearly discernible waveform by visual analysis of stacked waveforms from highest to lowest SPL. Amplitudes of waves I and V of the ABR in response to a stimulus level of 80 dB SPL were measured for analysis of the wave-V/I amplitude ratio. The wave amplitude was defined as the height of a peak in microvolts from the top of the peak to the lowest point of the following negative trough, which is known as the peak-to-peak amplitude.ABRs were measured in a sound booth  in each individual ear of blast-exposed rats and unexposed naïve control rats at four time points—i.e., prior to blast exposure and at 24 h, 4 wk and 8 wk after exposure. For testing, animals were anesthetized with ketamine (90 mg/kg) and xylazine (9 mg/kg) via intraperitoneal injection, while basal body temperature was maintained with a servo heating pad. Stimulus generation and data acquisition were accomplished with a RX6 workstation , running TDT BioSigRP software. Acoustic stimuli were tone bursts of alternating polarity, with 5-ms plateau and 0.5-ms coset al. (2006) and was based upon the ability of the acoustic startle response to be considerably reduced when preceded by a silent gap in a constant acoustic background [PPI tests were conducted in blast-exposed rats and in unexposed, age- and sex-matched naïve control rats before exposure and at 4- and 8-weeks after exposure. The PPI detection method was adapted from Turner ckground . The ratDuring the two-minute acclimation phase, animals adapted to the test environment . In block I, three startle pulse alone trials were run in quiet to habituate the startle response to a more stable baseline. No data were collected in this block. In block II, we used four different frequencies  to match tinnitus pitch. Thus, there were four different prepulse-pulse trials, each paired with a startle pulse alone trial to be displayed under each of the two prepulse conditions . A total of eight pairs of trials were pseudorandomized and presented 12 times, each throughout block II, which had 8×2×12 = 192 trials. The twelve data traces per trial type for the same pair were averaged, and the resulting value for the prepulse-pulse trials was divided by the startle pulse alone value. This quotient was the amount of PPI for each subject for each given frequency, where a value of 1 means no effect of the prepulse, a value of 0 means complete prepulse inhibition of the startle. In some PPI trials, however, absolute response amplitudes differed considerably between presentations of the same trial. As such, we applied Grubb’s test to determine if the most extreme value in a set of trials of the same type was an outlier by analysis of its Z-score  in the use of tinnitus index score as an assay for tinnitus [Tinnitus determination was based upon the method of Sametsky tinnitus . Tinnituhttp://www.masseyeandear.org/research/ent/eaton-peabody/epl-histologyresources/). Confocal z-stacks at the 2, 4, 8, 16, 32 and 48 kHz regions were obtained. Images were acquired using a Zeiss LSM-710 confocal microscope , in a 1024×1024-pixel frame with z steps of 0.5 μm. From an endolymphatic surface view of the organ of Corti, each stack contained ribbon synapses of six to nine IHCs. Image stacks were processed using Amira 3D software (FEI). All quantitative analyses were performed with raw image stacks. The presynaptic ribbons (red channel) were identified by segmentation, then quantified and tracked in the z-dimension to avoid superpositional ambiguity or overestimations in each stack. Individual ribbons were isolated, counted, and expressed as synaptic ribbons per IHC in the stack.Cochlear tissue collection and ribbon synapse counting have been described previously . In briepost hoc test (Tukey) were used to determine if there were statistically-significant differences among groups and between each of two groups. P values of less than 0.05 were regarded as statistically-significant.For quantitative biomarker analyses, one-way ANOVA (SPSS 14.0 for windows) and a F-statistic and leading to a more powerful ANOVA test. When performing a two-factor ANOVA with repeated measures in either one factor (a mixed ANOVA) or both factors (a two-way repeated measures ANOVA), we herein assumed that sphericity could hold, which is the requirement for ANOVA with matched samples. Note that if there are only two levels of repeated measures then sphericity is automatically satisfied. In this study, the factor “time” that was repeated measures had only two levels (4- and 8-weeks post-exposure), then there is no reason to be concerned about violations of sphericity. If a significant interaction of “frequency” with “group” (or “time”) was found in ANOVA, we examined the simple effect of “group” (or “time”) at each specified value of “frequency”. Simple effects were assessed for significance using the Holm-Bonferroni method. Here, binary logistic regression was employed to achieve two goals: (1) to determine whether there was a relationship between the amplitude ratio V/I of ABRs and the presence/absence of tinnitus, in hopes of understanding more about the mechanism of tinnitus; and (2) to find an equation that would predict the probability of tinnitus with a given ratio V/I, to help guide development of an objective measure for tinnitus. The fit of logistic regression model was tested using the following measures: likelihood ratio test, Wald test, Hosmer-Lemeshow test, classification table, ROC curve and pseudo R-squareds. Also, loess  analysis was used for fitting a smooth curve between the ABR wave-V/I ratio and either of ABR threshold shifts and cochlear synapse counts to reveal whether hearing loss and/or cochlear synaptopathy would affect the ratio of ABR wave V to wave I. Statistical analysis was performed using Real Statistics Using Excel  for logistic regression, R version 3.4.3 (http://www.r-project.org/) for loess curve fitting, and GraphPad Prism 4  for ANOVA, respectively.For mean tinnitus index scores, a confidence interval was constructed at a confidence level of 95% for each test frequency using data from the control group. The interval that we can be 95% certain contains the true mean index score in the normal population was applied as a diagnostic criterion for tinnitus. That is, blast-exposed subjects with scores falling above the 95% confidence interval of the companion control group were considered to have tinnitus at that given test frequency. Fisher’s exact test was used to evaluate whether the treatment and non-treatment groups would differ in proportion developing tinnitus. In most cases, two-factor ANOVAs with “frequency” × “group” (or “time”) were performed on datasets of our observed response variables  to find the differences between the control and exposure groups. Here, “group” was commonly referred to as a between-subjects factor, while “frequency” was always treated as a within-subjects factor  since our experimental design was so that we took multiple measures across frequencies per subject. When investigating changes in mean values over two or more time points, which were measured in the same exposure group, we would treat “time” as a within-subjects factor as well to partition out the variability due to the individual differences between subjects. This has the effect of increasing the value of the In previous studies of rats exposed to mild blast injuries, chronic neuronal spontaneous hyperactivity in central auditory nuclei has been reported as a conjunctive sequela associated with peripheral hearing loss –52. ThisTo examine whether we could detect objective molecular evidence of chronic blast-induced neuroplastic changes and whether these putative response patterns were addressable by NAC/HPN-07 treatment, we exposed rats to a single shock tube blast of 10 psi (~190 dB peak SPL), which consistently induced minor to moderate permanent ABR threshold shifts in untreated controls of 8.7±1.1, 8.0±1.4, 15.8±1.4, and 42.0±2.7 dB at 4, 8, 16, and 24 kHz, respectively, at eight weeks post-blast in our electrophysiological evaluation studies. Treatment with either vehicle  or NAC/HPN-07 was initiated at one-hour post-injury, and twice-daily treatments were repeated on two consecutive days thereafter. Animals were then allowed to recover for nine weeks prior to harvesting tissues for histopathological examination of chronic blast-induced changes.AR-α1), and glutamate receptor 2 (GluR2). Arc is a neuroplasticity protein that regulates synaptic strength by enhancing the endocytic trafficking rate of AMPA receptors and has been widely used as a tinnitus biomarker in the brain [arrows in arrows in arrows in arrows in Brain tissues from central auditory nuclei of the blast-exposed rats in each cohort were immunolabeled with antibodies directed against activity-regulated cytoskeleton-associated protein , gamma-aminobutyric acid-type A receptor alpha 1 , such that there was no significant difference between the naïve controls and the NAC/HPN-07 treatment groups in the AC and the IC (p > 0.05). Although there was a clear recovery trend for Arc immunoreactivity in the DCN in response to NAC/HPN-07 treatment, statistical significance could not be assigned relative to the vehicle-treated group in this auditory center , such that the percent increase (86%) closely mirrored the percent decrease in Arc-positive neurons in this auditory center , such that there was no significant difference between naïve controls and therapeutic-treated animals at nine weeks post-blast (p > 0.05). Taken together, these results suggest that chronic reductions in Arc levels in central auditory structures and corresponding AMPAR accumulation in the DCN of blast-exposed rats may predispose these auditory nuclei to stimulus-independent (spontaneous) activity.In blast-exposed rats treated with NAC/HPN-07, the number of GluR2-positive neurons in the DCN was significantly reduced compared to the vehicle-treated group . A similar, persistent elevation in the density of GABAAR-α1 immunoreactivity was observed throughout the AC at nine weeks post-blast , with 100% of animals in this cohort exhibiting significant elevations in VR1-positive immunostaining. As disproportionately-high VR1 activation in the cochlea has been shown to result in elevated baseline firing rates among SGNs, these results suggest that the SG in vehicle-treated animals might be susceptible to chronic synaptic dysregulation post-blast [In addition to central auditory pathway neuroplastic changes, chronic upregulation of vanilloid receptor 1  expression in cochlear spiral ganglion neurons (SGNs) has been reported as a peripheral biomarker for tinnitus-related hyperactivity induced by an acoustic trauma . Similarst-blast .p < 0.001 compared to vehicle-treated controls, one-way ANOVA with Tukey post-test, p > 0.05). These results indicate treatment-specific mitigation of prolonged blast-induced VR1 upregulation among neurons in the SG after antioxidant treatment.We also evaluated and quantified the relative density of strongly VR1-immunopositive SGNs in blast-exposed rats that were subsequently treated with NAC/HPN-07 . TherapeIn summary, blast-induced changes in key neuroplasticity-related biomarkers in the peripheral and central auditory pathways provided objective evidence that the shock tube blast model induced chronic imbalances in synaptic gating factors that have previously been observed in animals with behavioral manifestations of tinnitus. Moreover, these seemingly maladaptive changes were significantly mitigated by treatment with NAC/HPN-07 post-injury, suggesting that this therapeutic intervention might short-circuit the development of this auditory disorder in blast-exposed animals.To test this hypothesis, new cohorts of animals were exposed to the same blast overpressure model, and methods and measures were employed to evaluate behavioral evidence of tinnitus in blast-exposed animals treated with either vehicle or NAC/HPN-07. To this end, a stimulus level at 80 dB SPL for the ABR amplitude measurements was selected to highlight the response from the low-SR fibers  that havBased on prior studies, tinnitus and hyperacusis are common sequelae of acoustic overexposure and are often comorbid , 2. Gap-F = 4.508, p < 0.05) and a non-significant interaction of exposure group and test frequency  = 1.890, p > 0.05) for the vehicle-treated group, suggesting that blast overpressure caused gap-detection deficits across a wide frequency range in rats. In contrast, neither a main effect of exposure group  = 1.716, p > 0.05) or a two-way interaction of group and frequency  = 0.5104, p > 0.05) were found to be significant for the NAC/HPN-07 treatment group, indicating that the drug formulation was effective in preventing gap-detection failures due to acoustic trauma. With a 95% confidence interval for the average control tinnitus index score determined for each test frequency (which was applied as a diagnostic criterion for tinnitus), we identified 17 out of the 20 blast-exposed animals with behavioral evidence of tinnitus in the vehicle treatment group (p < 0.01).Overall, the index scores were significantly increased in the 9–24 kHz range in the vehicle treatment group  but remant group , represent group , where tp > 0.05) and (2) acoustic PPI did not differ significantly in either a main effect of exposure group  = 2.829, p > 0.05) or an interaction of exposure group and test frequency  = 1.273, p > 0.05) in a mixed ANOVA. From these results, it was concluded that our blast exposure model did not exaggerate startle-based behavior of possible relevance to hyperacusis.Hyperacusis was assessed with acoustic PPI, as well as with acoustic startle response (which was conducted in a quiet background). Here, blast-exposed rats in the vehicle-treated group showed control-like responses manifested both in acoustic startle amplitude  and in aF = 0.9747, p > 0.05) or in interaction of test frequency and exposure group  = 0.2852, p > 0.05). By means of repeated observations at four and eight weeks after exposure, subsequent within-group comparisons revealed that an ABR threshold shift could be reduced by 6 dB  in the therapeutic treatment group but by only up to 3.5 dB (observed at 8 kHz) in the vehicle treatment group. Given that ABR threshold detection is associated with varying stimulus levels in 5-dB steps, the test-retest variability might be expected to be within 5 dB. As such, an attenuation of 6 dB at threshold, rather than a 3.5-dB attenuation that fell within the 5 dB ABR variability, can be reasonably regarded as a practically-important improvement in hearing level, even though both results as an estimate of within-group differences were statistically significant at p < 0.05  ABR thresholds in each animal and compared to the average thresholds for age-matched naïve control animals. Blast-induced hearing loss is summarized in F = 28.89, p < 0.0001) and for ABR wave-V amplitude  = 22.48, p < 0.0001), culminating in the determination of a significant interaction for the wave-V/I ratio  = 6.910, p < 0.001). In addition, significant simple effects were determined for wave-I amplitude at 4, 16 and 24 kHz, for wave-V amplitude at 24 kHz, and for the wave-V/I ratio at 4, 16 and 24 kHz, relative to naïve controls. In NAC/HPN-07 treated rats, the ANOVA analysis revealed a significant interaction of group and frequency both for wave-I amplitude  = 29.42, p < 0.0001) and for wave-V amplitude  = 17.18, p < 0.0001), each with a significant simple effect of group on the amplitudes at 4, 16 and 24 kHz relative to naïve controls. However, for the ABR wave-V/I ratios in this treatment group, there were no calculated significant differences, either in interaction of group and frequency  = 1.576, p > 0.05) or in main effect of group on the amplitude ratio  = 0.3066, p > 0.05).The ABR wave-I and -V amplitudes and the resultant wave-V/I amplitude ratios in response to 80 dB SPL were obtained at each test frequency for each blast-exposed ear and then normalized with respect to the corresponding mean values among age-matched, naïve controls. The ABR wave I represents the summed activity of the spiral ganglion neurons (SGNs), while the ABR wave V, which is generated in the lateral lemniscus and inferior colliculus , reflectAs seen in Recent work shows that cochlear synaptopathy (known as the loss of synapses between SGNs and IHCs) may be crucial for the compensatory hyperactivity that drives gain in central auditory pathways , 29. It F = 4.2771 p < 0.01). In contrast, no significant change in wave-I amplitudes over this same time period post-blast was found at any test frequency in vehicle-treated animals  = 3.455, p < 0.05).As shown in  animals , though red) puncta, as illustrated in To more directly assess the potential for blast-induced cochlear synaptopathy and the positive therapeutic effect of NAC/HPN-07 on this pathophysiological response, we selected 12 ears from the naïve control group, 21 ears from the vehicle treatment group, and 23 ears from the NAC/HPN-07 treatment group for histological evaluation of IHC ribbon synapse densities. In maximum projections from confocal z-stacks, synaptic ribbons in IHCs appeared as pre-synaptic C-terminal binding protein 2 (CtBP2)-positive  = 4.590, p < 0.001), the simple effect of group proved to be significant at 32 and 48 kHz. For the comparison between NAC/HPN-07-treated ears and vehicle-treated ears, following a non-significant interaction  = 1.628, p > 0.05), the main effect of group collapsing across frequency turned out to be significant in a mixed ANOVA  = 10.20 p < 0.01). Taken together, the histological results demonstrated that NAC/HPN-07 had a positive treatment effect on cochlear synaptopathy. As shown in To confirm these observations, mean ribbon counts per IHC were computed, the results from which are shown in variables as well as capturing a trend in data that might be depicted by the slope of a trendline. Where, the slope of the V/I ratio with ribbon count was far from zero, the slope relating the V/I ratio to ABR threshold shift was near zero. Thus, the loess curves revealed a dependence of elevated ABR wave-V/I ratios on loss of IHC ribbon synapses, with no clear dependence on ABR threshold shift (due to OHC loss). Therefore, these results suggest that cochlear synaptopathy, rather than direct OHC damage, was key to the development of disproportionate activity changes in the blast-exposed rats.To determine whether an enhancement of the ABR wave-V/I ratio is ascribable to a reduction in synaptic ribbon counts and/or an elevation in cochlear ABR thresholds, we applied the loess method to model such a relationship; that is, determination of whether ribbon counts or threshold shifts might serve as a predictor variable in order to observe the effect on an outcome of the wave-V/I amplitude ratio. Here, the ABR wave V/I ratio and the number of IHC ribbons were averaged for frequencies from 4–32 kHz in each blast-exposed ear and were normalized to their respective naïve control means. This process aimed to ensure that the physiological and histological data could correspond to each other in the regression model, especially in terms of the frequency-specific effect on variables. The loess regression fitted a smooth curve through points in the scatter plot of the wave-V/I ratio vs. the ribbon count  or the tAccording to one widely-accepted view regarding the central tinnitus generator, tinnitus is considered to arise from a compensatory increase in gain  in the central auditory system in response to the loss of peripheral input from the cochlea , 81. In dichotomous variable with only two possible outcomes—yes and no (coded as 1 and 0)—measured in the PPI behavioral model, while “wave-V/I ratio” is a measurement variable whose value denotes the maximum value among 8 measurements  in each individual subject. p-values of 0.000759 (χ2(1) = 11.34) and 0.0151 (χ2(1) = 5.8947), respectively, suggesting that the ABR wave V/I amplitude ratio contributes significantly to the prediction of tinnitus. Also, as a measure of goodness of fit, the Hosmer–Lemeshow test showed a non-significant result (χ2(34) = 38.0693, p = 0.2893), providing evidence that the model was a good fit. The Hosmer–Lemeshow test is a formal test of the null hypothesis that the fitted model is correct, and its output is a p-value—a number between 0 and 1, with higher values  indicative of a better fit. Further checks of model fit were carried out on the model’s predictive power by inspection of statistics of classification table, ROC curve, pseudo-R2.In the present study, we used a binary logistic regression to find the best fitting model to determine the relationship between the presence/absence of tinnitus and the ABR wave V/I ratio, resulting in equation that can be used to predict the probability of a chronic tinnitus percept from the wave-V/I ratio. Here, “tinnitus” is a R2 measures, the model had a McFadden’s R2 of 0.24, a Cox & Snell’s R2 of 0.26 and a Nagelkerke’s R2 of 0.36, respectively. Basically, values of 0.2 to 0.4 for the McFadden’s R2 represent excellent model fit. In essence, all the statistics . RecentOur comparative analyses from the control and vehicle groups share similarities with these previous findings; namely, blast-induced cochlear neuropathy  and (2) measures of goodness of fit . The fit statistics Using this model of blast-induced tinnitus, we found that therapeutic intervention with NAC/HPN-07 significantly reduced the incidence of tinnitus  in the cNAC’s efficacy as a neuroprotectant is well-established , 83, 84.AR-α1 and GluR2 in the AC, IC and DCN. We observed that, in untreated animals, blast overpressures caused chronic downregulation of Arc in the central auditory pathway, which is consistent with prior studies on tinnitus [AR-α1 and GluR2 in these brain nuclei and elevated levels of VR1 in the SG.To explore molecular mechanisms of tinnitus, we surveyed the expression levels of neuroplasticity-related biomarkers: (1) VR1 in the SG and (2) Arc, GABAtinnitus , 54, 56,tinnitus . We alsoPrior work suggests that cochlear neuropathologic changes, such as loss of IHC synapses and changes in BDNF or c-Fos levels in the SG, trigger downregulation of Arc in central auditory pathways, which has been correlated with tinnitus , 54, 55.Under normal central conditions, salient stimuli up-regulate Arc activity in neurons to promote post-synaptic AMPA receptor endocytosis to govern and maintain synaptic strength , 90. TheARα-1 levels in the DCN and AC. We found that, while GABAAR-α1 immunolabeling was more prevalent in the DCN, this immunoreactivity pattern was restricted to GAD67-positive neurons Click here for additional data file.S1 Fig(TIF)Click here for additional data file.S1 Raw Images(PDF)Click here for additional data file.